Botto Margherita M, Murthy Sudarshan, Lamers Meindert H
Cell and Chemical Biology Department, Leiden University Medical Center, Einthovenweg 20, 2333 ZC Leiden, The Netherlands.
ACS Omega. 2023 Feb 20;8(9):8285-8292. doi: 10.1021/acsomega.2c06577. eCollection 2023 Mar 7.
Exonucleases are essential enzymes that remove nucleotides from free DNA ends during DNA replication, DNA repair, and telomere maintenance. Due to their essential role, they are potential targets for novel anticancer and antimicrobial drugs but have so far been little exploited. Here, we present a simple and versatile real-time exonuclease assay based on 2-aminopurine, an intrinsically fluorescent nucleotide that is quenched by neighboring bases when embedded in DNA. We show that our assay is applicable to different eukaryotic and bacterial exonucleases acting on both 3' and 5' DNA ends over a wide range of protein activities and suitable for a high-throughput inhibitor screening campaign. Using our assay, we discover a novel inhibitor of the PHP-exonuclease that is part of the replicative DNA polymerase DnaE1. Hence, our novel assay will be a useful tool for high-throughput screening for novel exonuclease inhibitors that may interfere with DNA replication or DNA maintenance.
核酸外切酶是在DNA复制、DNA修复和端粒维持过程中从游离DNA末端去除核苷酸的必需酶。由于它们的重要作用，它们是新型抗癌和抗菌药物的潜在靶点，但迄今为止很少被开发利用。在这里，我们提出了一种基于2-氨基嘌呤的简单通用的实时核酸外切酶检测方法，2-氨基嘌呤是一种内在荧光核苷酸，当嵌入DNA中时会被相邻碱基淬灭。我们表明，我们的检测方法适用于作用于3'和5'DNA末端的不同真核和细菌核酸外切酶，适用于广泛的蛋白质活性范围，并且适用于高通量抑制剂筛选活动。使用我们的检测方法，我们发现了一种新型的PHP核酸外切酶抑制剂，它是复制性DNA聚合酶DnaE1的一部分。因此，我们的新型检测方法将成为高通量筛选可能干扰DNA复制或DNA维持的新型核酸外切酶抑制剂的有用工具。
